Abstract
Notch signaling is involved in the early onset of osteoarthritis. The aim of this study was to investigate the role of Notch signaling changes during proliferation and differentiation of chondrocyte, and to testify the mechanism of MMP-13 regulation by Notch and Runx2 expression changes during osteoarthritis. In this study, Chondrocytes were isolated from rat knee cartilages. Notch signaling was activated/inhibited by Jagged-1/DAPT. Proliferative capacity of Chondrocytes was analyzed by CCK-8 staining and EdU labeling. ColX, Runx2 and MMP-13 expressions were analyzed as cell differentiation makers. Then, Runx2 gene expression was interfered using lentivirus transfection (RNAi) and was over-expressed by plasmids transfected siRNA in chondrocytes, and MMP-13 expression was analyzed after Jagged-1/DAPT treatment. In vivo, an intra-articular injection of shRunx2 lentivirus followed with Jagged1/DAPT treatments was performed in rats. MMP-13 expression in articular cartilage was detected by immunohistochemistry. Finally, MMP-13 expression changes were analyzed in chondrocytes under IL-1β stimulation. Our findings showed that, CCK-8 staining and EdU labeling revealed suppression of cell proliferation by Notch signaling activation after Jagged-1 treatment in chondrocytes. Promoted differentiation was also observed, characterized by increased expressions of Col X, MMP-13 and Runx2. Meanwhile, Sox9, aggrecan and Col II expressions were down-regulated. The opposite results were observed in Notch signaling inhibited cells by DAPT treatment. In addition, Runx2 RNAi significantly attenuated the ‘regulatory sensitivity’ of Notch signaling on MMP-13 expression both in vitro and in vivo. However, we found there wasn’t significant changes of this ‘regulatory sensitivity’ of Notch signaling after Runx2 over-expression. Under IL-1β circumstance, MMP-13 expression could be reduced by both DAPT treatment and Runx2 RNAi, while Runx2 interference also attenuated the ‘regulatory sensitivity’ of Notch in MMP-13 under IL-1β stimulation. In conclusion, Notch signaling is an important regulator on rat chondrocyte proliferation and differentiation, and this regulatory effect was partially mediated by proper Runx2 expression under both normal and IL-1β circumstances. In the meanwhile, DAPT treatment could effectively suppress expression of MMP-13 stimulated by IL-1 β.
Highlights
Osteoarthritis (OA) is the most common degenerative articular disorder nowadays[1]
Initial cleavage of Col II is preferentially performed by Matrix metalloproteinase 13 (MMP-13), an interstitial collagenase expressed by articular chondrocytes[5,6,7]
In early OA, the normally quiescent chondrocytes undergo phenotypic shift, characterized by a transient proliferative response, a differentiation expressing hypertrophy-like changes and increased synthesis of cartilage-degrading enzymes, such as MMP-1325,26
Summary
Osteoarthritis (OA) is the most common degenerative articular disorder nowadays[1]. Currently, there haven’t been effective interventions to prevent the initial onset or arrest the progression of OA, which is due to the limited understanding of molecular mechanisms in the progress of the disease. Loss of RBPjκ-dependent Notch signaling in postnatal joint cartilage results in an early OA-like pathology[18], while temporary suppression of Notch signaling in murine joints leads to delayed OA progression[19]. Shang et al.[20] recently found that there is a significantly positive correlation between Runx[2] expression, as well as MMP-13 expression, with Notch signaling in a chondrogenic cell line All these studies above led us to hypothesize that MMP-13 expression was possibly regulated by Notch signaling through activation of Runx[2] in chondrocytes, which regulatory mechanism might be required for the pathogenesis of early OA
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